The goal of our studies is to understand the biochemical pathways and to identify key proteins (enzymes) that are involved in the activation and the effector phase of such important immune responses, like T-cell- mediated cytotoxicity (toward virus-infected cells and toward tumor cells) and in lymphokine secretion (e.g. gamma-interferon and Interleukin 2). It is expected, that such knowledge will enable us to selectively manipulate different functional responses of T-cells with specifically designed immunomodulators. We attempt to reach this goal by taking advantage of the (A) extensive knowledge of the structure of well known protein kinases and by (B)developing new approaches for the isolation of novel, functionally important protein kinases. (A). Role of the cAMP-dependent protein kinase (PKA) in effector functions of T-helper and cytotoxic T-lymphocytes (CTL). The results obtained using molecular-biological approaches and synthetic peptide inhibitors suggest the novel view, that PKA has dual role in the regulation of T-lymphocytes effector functions; it down-regulates protein-synthesis independent responses (cytotoxicity and exocytosis of granules), but is required for the TCR-triggered, DNA transcription and protein synthesis-dependent processes (gamma interferon or IL-2 secretion). Thus PKA may serve as an attractive target for the immunomodulation. (B) Identification of novel ectoprotein kinases in lymphocytes. While pursuing our "extracellular ATP" hypothesis of the cell-mediated cytotoxicity we demonstrated the presence and characterized ecto ATPase(s) and highly active and extracellularly located protein kinases (ectokinases) which we implicated in the T-cell functions using several different inhibitors. These enzymes are unusual because they "look" outside the cell surface while residing in the plasma membrane of lymphocytes. At least two ectokinases with different substrate specificities are described to date: "Casein Kinase II-like" ectoenzyme that phosphorylates Ser/Thr in the negatively charged amino acid environment and "Histone-like" kinase with specificity toward basic amino acid surrounded Ser/Thr. These enzymes are now purified and attempts are being made to obtain the amino acid sequences of tryptic peptides to allow the cDNA cloning. Since we also identified PPIA class of ectoprotein phosphatases these data point on the presence of the complete phosphorylation/dephosphorylation system on the surface of lymphocytes.